Detection of Amines and Unprotected Amino Acids in Aqueous Conditions by Formation of Highly Fluorescent Iminium Ions

Article abstract of DOI:10.1021/ja036434m

Properly substituted coumarin aldehydes can be used to detect amines and amino acids under neutral, high ionic strength conditions by the formation of highly fluorescent iminium ions. The fluorescence of one sensor increases by 26-fold upon the addition o

Full text of DOI:10.1021/ja036434m

Published on Web 12/06/2003
Detection of Amines and Unprotected Amino Acids in Aqueous Conditions by
Formation of Highly Fluorescent Iminium Ions
Ellen K. Feuster and Timothy E. Glass*,†
Department of Chemistry, The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802
Received May 30, 2003; E-mail: teg@chem.psu.edu
Fluorescent probes have become a common means of investigat-
ing cellular events in real time via fluorescence microscopy. For
such applications, chemical sensors have proven to be invaluable,
particularly for tracking certain metal ions.1 We have been interested
in the development of practically useful chemical sensors for metal
ions and small organic compounds. We have reported progress
toward this goal using a structurally novel pinwheel receptor motif
which operates by cooperative interactions.2 As a potential sensor
substrate, amino acids are an attractive target, especially the
excitatory amino acids glutamate and aspartate.3 To adapt our
pinwheel receptors for recognition of amino acids, a good recogni-
tion element for the amine portion of the amino acid was required.
Aqueous phase recognition of amines by groups such as crown
ethers rely on the charged nature of the protonated form.4 Given
that amino acids are zwitterionic and overall neutral, these
functionalities are less useful for recognition of the amino group
of R-amino acids. Furthermore, ionic interactions are particularly
weakened in the high salt conditions necessary for cellular work.5
Thus, metal chelation has been employed to recognize unprotected
amino acids in aqueous conditions.6 Particularly noteworthy is
Anslyn’s aspartate sensor which uses dye displacement to produce
a chromophoric response.7
Figure 1. UV/visible absorption spectra for (a) compound 1a and (b)
compound 1b upon addition of glycine (10 µM in sensor with 100 mM
NaCl, 50 mM HEPES, pH ) 7.4, 37 °C).
Scheme 1
Taking a cue from the success of boronic acids in “binding”
diols via the formation of covalent boronate esters,8 we chose to
explore aldehyde containing chromophores to detect amines and
amino acids via the reversible formation of imines.9 The major
questions to be addressed in this study are: can imine formation
be used for amine recognition under neutral, high ionic strength
aqueous conditions, and can a system be designed which will
generate an appropriate fluorescent response upon imine formation?
The formation of imines of amino acids under aqueous conditions
has already been examined in detail in the context of understanding
the pyridoxal phosphate (PLP) dependent transamination.10 In fact,
the formation of PLP and related imines has been studied by
fluorescence.11,12 Our initial work focused on the fluorescent
behavior of several aldehyde substituted coumarins because of their
ready accessibility and desirable spectral properties. Imines of amino
acids have pKa’s above 7 and are largely protonated at neutral pH.13
Thus, coumarins such as 1 (Scheme 1) were particularly attractive
in that the iminium ion formed by reaction with an amine could
hydrogen bond to the carbonyl of the coumarin, thus effectively
modulating its fluorescence. Such fluorescence changes have been
observed upon metal chelation to properly designed coumarin
systems.14
remarkably distinct spectroscopic behavior upon the addition of
glycine (Figure 1). The equilibrium formation of the iminium ion
was poor for 1a (Keq < 1) as evidenced by a small red shift in the
absorption spectrum. In contrast, formation of the iminium ion was
substantially more favorable for 1b (Keq ) 4.0) and was ac-
companied by a much larger (34 nm) red shift in absorption. The
red shifted absorption that both compounds exhibited is consistent
with the proposed mechanism of hydrogen bonding of the iminium
ion to the chromophore carbonyl. Indeed, basifying a solution of
1b and saturating glycine to pH ) 10 shifted the visible band from
479 to 420 nm, consistent with a shift from iminium ion to imine.
NMR studies were then undertaken to elucidate the differences
between the two aldehydes. The coumarin aldehydes were not
sufficiently water soluble to study by NMR in pure D2O. Therefore,
the analogous water soluble compounds 3 and 4 were prepared.16
The spectral properties of compounds 3 and 4 were similar to the
corresponding coumarins (1a and 1b, respectively). Neither com-
pound exhibited evidence of hydrate in D2O, and both converted
cleanly to an equilibrium mixture of aldehyde and the corresponding
iminium upon addition of d7-glycine. Given the similarities in NMR
behavior, we attribute the dramatic difference in spectroscopic
properties of 1a and 1b to steric effects. Apparently, the electronic
effect of substituting an iminium ion for an aldehyde carbonyl has
a minimal effect on the absorption spectrum in both cases. In the
case of 1b, steric hindrance between the C4 substituent and the
iminium ion enforces the s-trans conformation which is favorable
for hydrogen bonding interactions with the chromophore carbonyl,
thereby producing a large change in absorbance. In water, hydrogen
The coumarin aldehyde 1a15 and its 4-butyl derivative 1b16 were
examined for their interactions with amino acids using UV/vis
spectroscopy under high salt aqueous conditions similar to those
found in minimal cell culture media (100 mM NaCl, 50 mM
HEPES, pH ) 7.4). In this media, the two aldehydes displayed
† Present address: Department of Chemistry, University of Missouri, Columbia,
MO 65211. E-mail: glasst@missouri.edu.
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J. AM. CHEM. SOC. 2003, 125, 16174-16175
10.1021/ja036434m CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
constants and larger fluorescence increases than the neutral amino
acids. Both of these observations are likely rooted in the higher
pKa of the ammonium groups and the resultant iminium ions derived
from these amino acids. Secondary amines and hydroxy acids did
not interact with the sensor (entries 10 and 11). Finally, in agreement
with the absorption data, compound 1a did not give significant
fluorescence changes upon addition of amines or amino acids
(entries 12 and 13).
These results suggest that properly substituted coumarin alde-
hydes are an excellent fluorogenic substrate for amines, operating
efficiently in high salt, neutral solution with excitation and emission
profiles similar to commercial fluorophores (e.g., BODIPY).
Furthermore, intramolecular hydrogen bonding in water can be used
to induce strong fluorescent responses to the binding of organic
compounds. Because of the ready accessibility of 4-substituted
coumarins, these chromophores should function as the fluorescent
read-out for amine containing compounds within a larger receptor
architecture. Work toward this goal is currently underway in our
laboratory.
Figure 2. Fluorescence spectra for compound 1b as a function of added
glycine (λex ) 495 nm, 10 µM in sensor with 100 mM NaCl, 50 mM
HEPES, pH ) 7.4, 37 °C).
Table 1. Equilibrium Constants and Maximum Fluorescence
Enhancements at 513 nm of Compounds 1a and 1b with Aminesa
entry
sensor
analyte
K
eq (M-1
)
Imax/I0b
Acknowledgment. This work was supported by the National
Institutes of Health (GM 59245, RR 10524).
1
2
3
4
5
6
7
8
9
10
11
12
13
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1a
1a
glycine
aspartate
glutamate
lysine
serine
4.0
2.3
2.4
6.5
5.2
2.5
1.4
6.7
12.5
-
26
40
45
29
23
23
29
15
22
-
Supporting Information Available: Experimental details (PDF).
This material is available free of charge via the Internet at
http://pubs.acs.org.
â-alanine
alanine
References
ethanolamine
1,3-diaminopropane
lactic acid
diethylamine
glycine
(1) (a) Chemosensors of Ion and Molecule Recognition; Desvergne, J. P.,
Czarnik, A. W., Eds.; NATO ASI Series C: 492; Kluwer Academic
Press: Dordrecht, 1997. (b) de Silva, A. P.; Gunaratne, H. Q. N.;
Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.; Radermacher, J. T.;
Rice, T. E. Chem. ReV. 1997, 97, 1515-1566.
(2) (a) Raker, J.; Glass, T. E. J. Org. Chem. 2002, 67, 6113-6116. (b) Raker,
J.; Glass, T. E. J. Org. Chem. 2001, 66, 6505-6512.
(3) Czarnik, A. W. Chem. Biol. 1995, 2, 423.
-
-
<1
<1
1.5c
2.5c
glutamate
a Measured by fluorescent titration of 1 with amines at 37 °C; λex
495 nm; 100 mM NaCl; 50 mM HEPES; pH ) 7.4. Imax is the maximum
intensity at saturation taken from the fit of the titration data. c Estimated
from the titration curve as the data could not be fit accurately.
)
(4) (a) Tsubacki, K.; Kusumoto, T.; Hayashi, N.; Nuruzzaman, M.; Fuji, K.
Org. Lett. 2002, 4, 2313-2316. (b) de Silva, A. P.; Sandanayake, K. R.
A. S. Angew. Chem., Int. Ed. Engl. 1990, 29, 1173-1175.
(5) (a) Rekharsky, M.; Inoue, Y.; Tobey, S.; Metzger, A.; Anslyn, E. J. Am.
Chem. Soc. 2002, 124, 14959-14967. (b) Hosaain, M. A.; Schneider,
H.-J. Chem.-Eur. J. 1999, 5, 1284-1290. (c) Hossain, M. A.; Schneider,
H.-J. J. Am. Chem. Soc. 1998, 120, 11208-9.
b
bonds are not expected to be very strong, and thus the iminium
ion derived from compound 1a can adopt conformations in which
the chromophore carbonyl is not hydrogen bonded, giving a very
small response.
(6) (a) Imai, H.; Misawa, K.; Munakata, H.; Uemori, Y. Chem. Lett. 2001,
688-689. (b) Mizutani, T.; Wada, K.; Kitagawa, S. J. Am. Chem. Soc.
1999, 121, 11425-11431.
(7) A¨ıt-Haddou, H.; Wiskur, S. L.; Lynch, V. M.; Anslyn, E. V. J. Am. Chem.
Soc. 2001, 123, 11296-11297.
(8) (a) James, T. D.; Shinkai, S. Top. Curr. Chem. 2002, 218, 159-200. (b)
James, T. D.; Sandanayake, S.; Shinkai, S. Angew. Chem., Int. Ed. Engl.
1996, 35, 1910-1922.
(9) (a) Mertz, E.; Zimmerman, S. C. J. Am. Chem. Soc. 2003, 125, 3424-
3425. (b) Mertz, E.; Bell, J. B.; Zimmerman, S. C. Org. Lett. 2003, 5,
3127-3130.
(10) (a) French, T. C.; Bruice, T. C. Biochemistry 1964, 3, 1589-1596. (b)
French, T. C.; Auld, D. S.; Bruice, T. C. Biochemistry 1965, 4, 77-84.
(11) (a) Cambro´n, G.; Sevilla, J. M.; Pineda, T.; Bla´zquez, M. J. Flouresc.
1996, 6, 1. (b) Bla´zqez, M.; Ineda, T.; Sevilla, J. M.; Dom´ımguez, M.;
Garc´ıa-Blanco, F. J. Phys. Org. Chem. 1991, 4, 372. (c) See also: Vargas,
V.; Amigo, L. J. Chem. Soc., Perkin Trans. 2 2001, 1124.
(12) Fluorescent imines of amino acids have been studied under nonaqueous
conditions: Hwang, T. K.; Miller, J. N.; Burns, D. T. Anal. Chim. Acta
1978, 99, 305-315.
Fluorescence studies of compound 1b were then performed using
a variety of small molecule amines. By exciting in a region where
the iminium ion preferentially absorbs (495 nm), the sensor
produced a strong increase in fluorescence upon addition of an
amine. For example, upon titration with glycine, the fluorescence
intensity at 513 nm increased 26-fold (Figure 2). Results from a
number of amines and amino acids (Table 1) indicate that all
primary amines give strong fluorescence responses with compound
1b. The acidic amino acids (entries 2 and 3) had smaller equilibrium
(13) Kayser, R. H.; Pollack, R. M. J. Am. Chem. Soc. 1977, 99, 3379-3387.
(14) (a) Chen, C.-T.; Huang, W.-P. J. Am. Chem. Soc. 2002, 124, 6246-6248.
(b) Leray, I.; Lefevre, J.-P.; Delouis, J.-F.; Delaire, J.; Valeur, B. Chem.-
Eur. J. 2001, 7, 4590-4598 and references therein.
(15) Takechi, H.; Oda, Y.; Nishizono, N.; Oda, K.; Machida, M. Chem. Pharm.
Bull. 2000, 48, 1702-1710.
(16) See Supporting Information for synthesis and characterization.
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